EP3212666A1 - Human monoclonal antibody specific for the f protein of respiratory syncytial virus (rsv) - Google Patents
Human monoclonal antibody specific for the f protein of respiratory syncytial virus (rsv)Info
- Publication number
- EP3212666A1 EP3212666A1 EP15853881.9A EP15853881A EP3212666A1 EP 3212666 A1 EP3212666 A1 EP 3212666A1 EP 15853881 A EP15853881 A EP 15853881A EP 3212666 A1 EP3212666 A1 EP 3212666A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- seq
- antibody
- rsv
- amino acid
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000725643 Respiratory syncytial virus Species 0.000 title description 185
- 102000004169 proteins and genes Human genes 0.000 title description 10
- 108090000623 proteins and genes Proteins 0.000 title description 10
- 229960000402 palivizumab Drugs 0.000 claims abstract description 164
- 108010068327 4-hydroxyphenylpyruvate dioxygenase Proteins 0.000 claims abstract description 77
- 230000000890 antigenic effect Effects 0.000 claims abstract description 44
- 239000012634 fragment Substances 0.000 claims abstract description 43
- 238000000034 method Methods 0.000 claims abstract description 40
- 208000015181 infectious disease Diseases 0.000 claims abstract description 25
- 108010021625 Immunoglobulin Fragments Proteins 0.000 claims description 181
- 102000008394 Immunoglobulin Fragments Human genes 0.000 claims description 181
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 151
- 210000004027 cell Anatomy 0.000 claims description 99
- 239000002773 nucleotide Substances 0.000 claims description 79
- 125000003729 nucleotide group Chemical group 0.000 claims description 79
- 239000002299 complementary DNA Substances 0.000 claims description 62
- 108010047041 Complementarity Determining Regions Proteins 0.000 claims description 50
- 238000006386 neutralization reaction Methods 0.000 claims description 44
- 206010061603 Respiratory syncytial virus infection Diseases 0.000 claims description 18
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 12
- 102100026120 IgG receptor FcRn large subunit p51 Human genes 0.000 claims description 11
- 101710177940 IgG receptor FcRn large subunit p51 Proteins 0.000 claims description 9
- 108060003951 Immunoglobulin Proteins 0.000 claims description 9
- 102000018358 immunoglobulin Human genes 0.000 claims description 9
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims description 8
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 claims description 8
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 claims description 8
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 8
- 239000013604 expression vector Substances 0.000 claims description 7
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 6
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 6
- 239000003085 diluting agent Substances 0.000 claims description 6
- 210000002919 epithelial cell Anatomy 0.000 claims description 5
- 239000003937 drug carrier Substances 0.000 claims description 4
- 210000004962 mammalian cell Anatomy 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 2
- 230000003472 neutralizing effect Effects 0.000 abstract description 30
- 150000007523 nucleic acids Chemical class 0.000 abstract description 18
- 108020004707 nucleic acids Proteins 0.000 abstract description 17
- 102000039446 nucleic acids Human genes 0.000 abstract description 17
- 108020001507 fusion proteins Proteins 0.000 abstract description 4
- 102000037865 fusion proteins Human genes 0.000 abstract description 4
- 230000027455 binding Effects 0.000 description 54
- 235000001014 amino acid Nutrition 0.000 description 36
- 150000001413 amino acids Chemical class 0.000 description 35
- 229940024606 amino acid Drugs 0.000 description 33
- 241000700605 Viruses Species 0.000 description 24
- 241000196324 Embryophyta Species 0.000 description 22
- 239000000427 antigen Substances 0.000 description 20
- 230000035772 mutation Effects 0.000 description 19
- 102000036639 antigens Human genes 0.000 description 18
- 108091007433 antigens Proteins 0.000 description 18
- 238000002965 ELISA Methods 0.000 description 15
- 210000002966 serum Anatomy 0.000 description 15
- 238000000692 Student's t-test Methods 0.000 description 14
- 210000004072 lung Anatomy 0.000 description 14
- 239000000203 mixture Substances 0.000 description 14
- 239000012528 membrane Substances 0.000 description 13
- 238000003556 assay Methods 0.000 description 12
- 238000004587 chromatography analysis Methods 0.000 description 12
- 230000000694 effects Effects 0.000 description 12
- 238000004519 manufacturing process Methods 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 12
- 239000013598 vector Substances 0.000 description 12
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 11
- 229960001521 motavizumab Drugs 0.000 description 11
- 230000002829 reductive effect Effects 0.000 description 11
- 206010057190 Respiratory tract infections Diseases 0.000 description 10
- 210000003719 b-lymphocyte Anatomy 0.000 description 10
- 238000001514 detection method Methods 0.000 description 10
- 230000004927 fusion Effects 0.000 description 10
- 238000011282 treatment Methods 0.000 description 10
- 238000010790 dilution Methods 0.000 description 9
- 239000012895 dilution Substances 0.000 description 9
- 108090000765 processed proteins & peptides Proteins 0.000 description 9
- 235000018102 proteins Nutrition 0.000 description 9
- 230000009467 reduction Effects 0.000 description 9
- 238000005259 measurement Methods 0.000 description 8
- 238000002962 plaque-reduction assay Methods 0.000 description 8
- 241000144282 Sigmodon Species 0.000 description 7
- 230000037396 body weight Effects 0.000 description 7
- 230000003993 interaction Effects 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 230000003612 virological effect Effects 0.000 description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- 239000012894 fetal calf serum Substances 0.000 description 6
- 239000012530 fluid Substances 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 230000004048 modification Effects 0.000 description 6
- 238000012986 modification Methods 0.000 description 6
- 239000002245 particle Substances 0.000 description 6
- 238000007619 statistical method Methods 0.000 description 6
- 210000003501 vero cell Anatomy 0.000 description 6
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 5
- 241000282412 Homo Species 0.000 description 5
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 5
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 5
- 239000004472 Lysine Substances 0.000 description 5
- 241000144290 Sigmodon hispidus Species 0.000 description 5
- 235000009582 asparagine Nutrition 0.000 description 5
- 229960001230 asparagine Drugs 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 230000013595 glycosylation Effects 0.000 description 5
- 238000006206 glycosylation reaction Methods 0.000 description 5
- 230000036039 immunity Effects 0.000 description 5
- 210000004698 lymphocyte Anatomy 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 208000024891 symptom Diseases 0.000 description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 206010020751 Hypersensitivity Diseases 0.000 description 4
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 4
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 4
- 241000207746 Nicotiana benthamiana Species 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 4
- 230000001143 conditioned effect Effects 0.000 description 4
- 239000012228 culture supernatant Substances 0.000 description 4
- 238000010494 dissociation reaction Methods 0.000 description 4
- 230000005593 dissociations Effects 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 210000000987 immune system Anatomy 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000011533 pre-incubation Methods 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000011552 rat model Methods 0.000 description 4
- 238000003757 reverse transcription PCR Methods 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 238000012286 ELISA Assay Methods 0.000 description 3
- 241000711920 Human orthopneumovirus Species 0.000 description 3
- 206010061598 Immunodeficiency Diseases 0.000 description 3
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 3
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 3
- 206010024971 Lower respiratory tract infections Diseases 0.000 description 3
- 241000208125 Nicotiana Species 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 206010046306 Upper respiratory tract infection Diseases 0.000 description 3
- 235000004279 alanine Nutrition 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 3
- 235000018417 cysteine Nutrition 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 3
- 235000004554 glutamine Nutrition 0.000 description 3
- 210000002865 immune cell Anatomy 0.000 description 3
- 238000007918 intramuscular administration Methods 0.000 description 3
- 238000001990 intravenous administration Methods 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 229930182817 methionine Natural products 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 239000008194 pharmaceutical composition Substances 0.000 description 3
- 230000007505 plaque formation Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000000069 prophylactic effect Effects 0.000 description 3
- 239000012562 protein A resin Substances 0.000 description 3
- 208000020029 respiratory tract infectious disease Diseases 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 3
- 235000002374 tyrosine Nutrition 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 2
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 241000283984 Rodentia Species 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical group O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000006838 adverse reaction Effects 0.000 description 2
- 208000003455 anaphylaxis Diseases 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 235000003704 aspartic acid Nutrition 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 229930195712 glutamate Natural products 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- 210000004408 hybridoma Anatomy 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000016784 immunoglobulin production Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000001524 infective effect Effects 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000003456 ion exchange resin Substances 0.000 description 2
- 229920003303 ion-exchange polymer Polymers 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 229960000310 isoleucine Drugs 0.000 description 2
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 210000003097 mucus Anatomy 0.000 description 2
- 108010068617 neonatal Fc receptor Proteins 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- PHEDXBVPIONUQT-RGYGYFBISA-N phorbol 13-acetate 12-myristate Chemical compound C([C@]1(O)C(=O)C(C)=C[C@H]1[C@@]1(O)[C@H](C)[C@H]2OC(=O)CCCCCCCCCCCCC)C(CO)=C[C@H]1[C@H]1[C@]2(OC(C)=O)C1(C)C PHEDXBVPIONUQT-RGYGYFBISA-N 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 238000011321 prophylaxis Methods 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 239000012146 running buffer Substances 0.000 description 2
- 238000007480 sanger sequencing Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000013207 serial dilution Methods 0.000 description 2
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical group CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000002255 vaccination Methods 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 239000004474 valine Substances 0.000 description 2
- WGJUFIXHTBAMBX-BYPYZUCNSA-N (2s)-2,6,6-triaminohexanoic acid Chemical group NC(N)CCC[C@H](N)C(O)=O WGJUFIXHTBAMBX-BYPYZUCNSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 241000589158 Agrobacterium Species 0.000 description 1
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 1
- 206010002198 Anaphylactic reaction Diseases 0.000 description 1
- 206010002199 Anaphylactic shock Diseases 0.000 description 1
- 240000003291 Armoracia rusticana Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 206010007559 Cardiac failure congestive Diseases 0.000 description 1
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 description 1
- 208000002330 Congenital Heart Defects Diseases 0.000 description 1
- 201000003883 Cystic fibrosis Diseases 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241001115402 Ebolavirus Species 0.000 description 1
- 238000000729 Fisher's exact test Methods 0.000 description 1
- 108010019236 Fucosyltransferases Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 208000029462 Immunodeficiency disease Diseases 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- 208000019693 Lung disease Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- UEZVMMHDMIWARA-UHFFFAOYSA-N Metaphosphoric acid Chemical compound OP(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-N 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108010067902 Peptide Library Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 101710118538 Protease Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- IWUCXVSUMQZMFG-AFCXAGJDSA-N Ribavirin Chemical compound N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IWUCXVSUMQZMFG-AFCXAGJDSA-N 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 108010065282 UDP xylose-protein xylosyltransferase Proteins 0.000 description 1
- 108010059722 Viral Fusion Proteins Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 230000010530 Virus Neutralization Effects 0.000 description 1
- 102000010199 Xylosyltransferases Human genes 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 230000036783 anaphylactic response Effects 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 230000009830 antibody antigen interaction Effects 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 229940009098 aspartate Drugs 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 208000028831 congenital heart disease Diseases 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 230000000120 cytopathologic effect Effects 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 238000011157 data evaluation Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000022811 deglycosylation Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- 150000002019 disulfides Chemical group 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 210000001163 endosome Anatomy 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000033581 fucosylation Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 150000004676 glycans Chemical group 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 102000053391 human F Human genes 0.000 description 1
- 108700031895 human F Proteins 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000007813 immunodeficiency Effects 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000002642 intravenous therapy Methods 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 238000012933 kinetic analysis Methods 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000034217 membrane fusion Effects 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000002741 palatine tonsil Anatomy 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 238000005498 polishing Methods 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 229960000329 ribavirin Drugs 0.000 description 1
- HZCAHMRRMINHDJ-DBRKOABJSA-N ribavirin Natural products O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1N=CN=C1 HZCAHMRRMINHDJ-DBRKOABJSA-N 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000012453 solvate Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 229940036185 synagis Drugs 0.000 description 1
- 238000011191 terminal modification Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 239000013026 undiluted sample Substances 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Definitions
- This invention relates to an monoclonal antibody construct or antibody fragment specific for the fusion protein (F protein) of respiratory syncytial virus (RSV), nucleic acids encoding the antibody construct or fragment, and cell lines expressing the antibody construct or fragment.
- This invention further relates to methods for producing said monoclonal antibody construct or antibody fragment and to the use of said monoclonal antibody construct or antibody fragment for treating or preventing infection by RSV having a normal or mutated version of F protein.
- F protein mediates fusion of the viral membrane with the target cell membrane and thus mediates virus genome entry into the target cell.
- F protein embedded into the membrane of infected cells mediates fusion between infected cells and their neighbors, leading to the syncytia formation characteristic of RSV infection.
- F protein is a type I fusion protein that rearranges from a metastable pre-fusion conformation to a highly stable post-fusion structure. This structural change in the protein is necessary for membrane fusion. Antibody binding to antigenic site II/ A interferes with this structural change, thus preventing infection of the target cell as well as the fusion of infected cells with neighboring cells and the subsequent formation of syncytia.
- a detailed topological and operational map of epitopes involved in neutralization and fusion was constructed using rodent-derived neutralizing antibodies specific for F protein of RSV A2 strain. Three non-overlapping antigenic sites (A, B, and C) and one bridge site (AB) were identified.
- the commercially available anti-RSV monoclonal antibody, palivizumab binds to a highly conserved region on the extracellular domain of mature F protein, referred to as antigenic site II or site A (antigenic site II/ A), which is said to encompass amino acids 262 to 275.
- palivizumab validates the antigenic site II/A as a crucial and effective target for a monoclonal antibody for use as a prophylactic measure to prevent infections. Nonetheless, a subset of RSV strains are resistant to palivizumab, and use of palivizumab can select for such resistant strains.
- This invention is predicated, in part, on the discovery that fully human antibody constructs that recognize the F protein of RSV are able to neutralize both palivizumab-resistant and palivizumab-sensitive RSV strains. Further, methods and compositions are presented to further enhance the pharmacokinetics and cytotoxicity of the antibodies.
- All RSV strains that exhibit resistance to palivizumab have been shown to contain amino acid changes within a specific region in the antigenic region WA on the F protein (Zhao, X et al., 2004).
- palivizumab-resistant RSV strains selected in vitro had mutations at position 272 of the fusion protein, from lysine to asparagine, methionine, threonine, glutamine, or glutamate. Variants containing mutations at positions 272 and 275 were detected in breakthrough patients.
- antibodies targeting the same antigenic region might exhibit improved efficacy as compared to palivizumab and motavizumab in cases where mutations are found in these amino acid positions. Therefore, an urgent need exists to develop novel monoclonal antibodies that bind to the antigenic region 11/ A of RSV F protein with higher affinity than palivizumab and that are capable of preventing and/or treating infection by palivizumab-resistant RSV strains.
- Antibodies generated by the human immune system against pathogens such as RSV inherently exhibit lower propensity to react with human self-antigens.
- the novel antibody constructs or antibody fragments targeting RSV described herein are entirely constructed from human origin.
- Palivizumab is not sufficient to prevent infection by every RSV isolate.
- the antibody construct or antibody fragment preferably recognizes both palivizumab- sensitive and palivizumab-resistant RSV strains.
- the antibody construct or antibody fragment preferably has an affinity constant of greater than about 10 ⁇ 9 M, and preferably greater than about 10 ⁇ 10 M, or greater than about 10 ⁇ n M.
- the antibody construct or antibody fragment recognizes and neutralizes human RSV strains of type A and B.
- the antibody construct or antibody fragment recognizes and neutralizes more than one RSV strain, and preferably one or more palivizumab-resistant RSV strains.
- a fully human antibody construct or fragment thereof with specificity to RSV antigenic region WA and an affinity constant of at least 10 ⁇ 9 M, and preferably greater than 10 ⁇ 10 M is described.
- the fully human antibody construct or fragment thereof is capable of neutralizing RSV virus strains of type A and B.
- the antibody is capable of inhibiting binding by palivizumab.
- the antibody construct or antibody fragment neutralizes one or more palivizumab-resistant strains.
- the invention relates to a cell comprising one or more cDNA sequences which encode a heavy chain variable region and/or a light chain variable region, wherein each cDNA sequence is constructed from an RSV-infected human, which cell produces an antibody construct or antibody fragment comprising the heavy chain variable region and/or the light chain variable region, such that the antibody construct or fragment thereof binds to RSV antigenic region 11/ A.
- the antibody construct or antibody fragment binds to RSV antigenic region 11/ A with an affinity of greater than 1 x 10 ⁇ 9 .
- the cell is a eukaryotic cell.
- the cell is a mammalian cell.
- the cell is a plant cell.
- the cell is a HEK293T cell.
- the cell is a tobacco cell.
- the heavy chain variable region cDNA sequence is coupled to a cDNA sequence which encodes a constant region of human immunoglobulin.
- the constant region cDNA sequence is from a different patient than the heavy chain variable region and/or the light chain variable region.
- the human immunoglobulin is from IgGl.
- the heavy chain variable region cDNA sequence comprises a nucleotide sequence having at least 90% sequence homology to the nucleotide sequence of SEQ ID NO. : 2.
- the nucleotide sequence encoding a complementarity determining region (CDR)l of the heavy chain of the antibody construct or antibody fragment comprises a nucleotide sequence having at least 90% sequence homology to the nucleotide sequence of SEQ ID NO.: 5.
- the nucleotide sequence encoding CDR2 of the heavy chain of the antibody construct or antibody fragment comprises a nucleotide sequence having at least 90% sequence homology to the nucleotide sequence of SEQ ID NO.: 6.
- the nucleotide sequence encoding CDR3 of the heavy chain of the antibody construct or antibody fragment comprises a nucleotide sequence having at least 90% sequence homology to the nucleotide sequence of SEQ ID NO. : 7.
- the light chain variable region cDNA sequence is coupled to a cDNA sequence which encodes a light chain constant region of human immunoglobulin.
- the constant region cDNA sequence is from a different patient than the light chain variable region and/or the heavy chain variable region.
- the human immunoglobulin is from IgGl.
- the light chain variable region cDNA sequence comprises a nucleotide sequence having at least 90% sequence homology to the nucleotide sequence of SEQ ID NO. : 4.
- the nucleotide sequence encoding CDR1 of the light chain of the antibody construct or antibody fragment comprises a nucleotide sequence having at least 90% sequence homology to the nucleotide sequence of SEQ ID NO.: 11.
- the nucleotide sequence encoding CDR2 of the light chain of the antibody construct or antibody fragment comprises a nucleotide sequence having at least 90% sequence homology to the nucleotide sequence of SEQ ID NO.: 12.
- the nucleotide sequence encoding CDR3 of the light chain of the antibody construct or antibody fragment comprises a nucleotide sequence having at least 90% sequence homology to the nucleotide sequence of SEQ ID NO.: 13.
- this invention relates to a cDNA sequence as described herein. In one embodiment, this invention relates to an RNA molecule encoded by a cDNA sequence as described herein.
- this invention is directed to an antibody construct or antibody fragment that binds to RSV antigenic region WA.
- the antibody construct or antibody fragment binds to RSV antigenic region II/ A with an affinity of greater than 1 x 10 "9 M.
- the antibody construct or antibody fragment is capable of neutralizing at least one RSV strain.
- the antibody construct or antibody fragment is capable of neutralizing one or more RSV strains that are resistant to palivizumab.
- the neutralization capacity of the antibody construct or antibody fragment against at least one RSV strain is at least 2 times greater than the neutralization capacity of palivizumab.
- the heavy chain variable region of the antibody construct or antibody fragment comprises an amino acid sequence having at least 90% sequence homology to the amino acid sequence of SEQ ID NO.: 1.
- the amino acid sequence of CDR1 of the heavy chain of the antibody construct or antibody fragment comprises an amino acid sequence having at least 90% sequence homology to GASINLYD (SEQ ID NO.: 8).
- the amino acid sequence of CDR2 of the heavy chain of the antibody construct or antibody fragment comprises an amino acid sequence having at least 90% sequence homology to GYISGST (SEQ ID NO.: 9).
- the amino acid sequence of CDR3 of the heavy chain of the antibody construct or antibody fragment comprises an amino acid sequence having at least 90% sequence homology to ARDVGWGPQYYYGLDV (SEQ ID NO.: 10).
- the antibody construct or antibody fragment recognizes an epitope on the F protein of RSV. In one embodiment, the antibody construct or antibody fragment recognizes an epitope within the antigenic region II/ A of the RSV F protein. In one embodiment, the antibody construct or antibody fragment recognizes an amino acid sequence having at least 90% sequence homology to the epitope of SEQ ID NO.: 23 and/or SEQ ID NO.: 24.
- the antibody construct or fragment is fully human. In one embodiment, the antibody construct or antibody fragment is chimeric. In one embodiment, the antibody construct or antibody fragment is humanized.
- the antibody construct or antibody fragment comprises
- a heavy chain complementarity determining region comprising the amino acid sequence GASINLYD (SEQ ID NO.: 8);
- a heavy chain CDR2 comprising the amino acid sequence GYISGST (SEQ ID NO.: 9);
- a heavy chain CDR3 comprising the amino acid sequence ARDVGWGPQYYYGLDV (SEQ ID NO.: 10);
- a light chain CDR1 comprising the amino acid sequence HSVQSTS (SEQ ID NO.: 14),
- a light chain CDR2 comprising the amino acid sequence GGS (SEQ ID NO.: 15), and
- a light chain CDR3 comprising the amino acid sequence QQSDRSPPIT (SEQ ID NO.: 16).
- the antibody or antibody fragment is constructed to comprise glycans lacking fucose and xylose, and/or incorporate an Fc region adapted to bind to neonatal receptor FcRn of epithelial cells with at least 10-fold (or 100- fold) less affinity than AR201.
- the antibody is a modified version of AR201, e.g., retaining the 6 CDRs identified herein.
- this invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising an antibody construct or antibody fragment as described herein and a pharmaceutically acceptable carrier, diluent, or excipient.
- the antibody construct or antibody fragment is lyophilized.
- the antibody construct or antibody fragment is in an aqueous solution.
- this invention relates to a bag for intravenous therapy, comprising the antibody construct or antibody fragment as described herein and a pharmaceutically acceptable carrier, diluent, or excipient.
- this invention relates to a method of producing an antibody construct or functional part thereof, the method comprising culturing a cell as described above under conditions in which the cDNA sequences are expressed, thereby producing an antibody construct or fragment that binds with specificity to RSV antigenic region WA.
- the antibody or a fragment thereof bind to RSV antigenic region II/A with an affinity of greater than 1 x 10 "9 M.
- the antibody or fragment thereof is capable of neutralizing at least one RSV strain.
- the antibody or fragment thereof is capable of neutralizing one or more RSV strains that are resistant to palivizumab.
- this invention relates to a chromatography column or membrane comprising an antibody construct or antibody fragment as described herein, wherein the antibody construct or antibody fragment is bound to the chromatography column or membrane.
- the chromatography column or membrane comprises Protein A, e.g. a Protein A resin.
- the chromatography column or membrane comprises an ion exchange resin.
- the chromatography column or membrane comprises a hydrophobic charge induction chromatography column.
- this invention relates to a method for treating a patient infected with RSV by administering an antibody construct or antibody fragment as described herein to the patient.
- this invention relates to a method for preventing infection by RSV in a patient at risk for RSV infection by administering an antibody construct or antibody fragment as described herein to the patient. Administration may be by any suitable method, as determined by a skilled clinician.
- the antibody construct or antibody fragment is administered by intramuscular or intravenous
- Figure 1 is a schematic of the process used to isolate an antibody specific for
- Figure 2 describes the complete nucleotide sequence (SEQ ID NO.: 1) and the amino acid sequence (SEQ ID NO.: 2) of the variable region of the heavy chain of AR201.
- Figure 3 describes the complete nucleotide sequence (SEQ ID NO.: 3) and the amino acid sequence (SEQ ID NO.: 4) of the variable region of the kappa light chain of AR201.
- Figure 4 describes the complete nucleotide sequences (SEQ ID NO.: 5; SEQ ID NO.: 5; SEQ ID NO.: 5; SEQ ID NO.: 5; SEQ ID NO.: 5; SEQ ID NO.: 5; SEQ ID NO.: 5; SEQ ID NO.: 5; SEQ ID NO.: 5; SEQ ID NO.: 5; SEQ ID NO.: 5; SEQ ID NO.: 5; SEQ ID NO. 5; SEQ ID NO. 5; SEQ ID NO. 5; SEQ ID NO. 5; SEQ ID NO. 5; SEQ ID NO. 5; SEQ ID NO.: 5; SEQ ID NO.: 5; SEQ ID NO.: 5; SEQ ID NO.: 5; SEQ ID NO.: 5; SEQ ID NO.: 5; SEQ ID NO.: 5; SEQ ID NO.: 5; SEQ ID NO.: 5;
- Figure 5 describes the complete nucleotide sequences (SEQ ID NO.: 11;
- Figure 6 describes the binding of purified AR201 ( ⁇ ) and human nonspecific monoclonal IgGl ( A ) to RSV-EIA antigen over a broad range of concentrations.
- Figure 7 describes the recognition and binding of reference strains of RSV-
- Figures 8A and 8B compares the recognition of clinical isolates of RSV by
- AR201 black bars
- palivizumab white bars
- Figure 8C indicates the ratio of binding of AR201 and palivizumab to clinical isolates of RSV. Black bars indicate a binding ratio of greater than 5 for the binding of AR201 over palivizumab to the respective clinical RSV isolate, indicative of resistance of the clinical isolate towards palivizumab.
- Figure 9A provides the nucleotide sequence (SEQ ID NO.: 17) of the F protein of a clinical isolate that is resistant to palivizumab.
- Figure 9B describes the amino acid sequence (SEQ ID NO.: 18) of the F protein of one clinical isolate resistant to palivizumab.
- Figure 10 provides the amino acid sequence of the palivizumab epitope region (SEQ ID NO.: 23), and the corresponding amino acid sequence of the F protein of a palivizumab-resistant strain (SEQ ID NO.: 24) that is recognized by AR201.
- Figure 11 provides the predicted peptides (SEQ ID NO. : 25 and SEQ ID NO.
- Figure 12 describes Asp-N cleavage of RSV F protein that is bound by
- AR201 black bars
- palivizumab white bars
- compositions and methods include the recited elements, but not excluding others.
- Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed invention.
- Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.
- composition refers to a composition that is suitable for administration to a human.
- Such compositions include various excipients, diluents, carriers, and such other inactive agents well known to the skilled artisan.
- Therapeutically effective amount refers to an amount of a drug or an agent that, when administered to a patient suffering from a condition, will have the intended therapeutic effect, e.g. , alleviation, amelioration, palliation or elimination of one or more manifestations of the condition in the patient.
- the therapeutically effective amount will vary depending upon the patient and the condition being treated, the weight and age of the subject, the severity of the condition, the salt, solvate, or derivative of the active drug portion chosen, the particular composition or excipient chosen, the dosing regimen to be followed, timing of administration, the manner of administration and the like, all of which can be determined readily by one of ordinary skill in the art.
- the full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a
- therapeutically effective amount may be administered in one or more administrations.
- Treatment covers the treatment of a patient, and includes: (a) reducing the risk of occurrence of the condition in a patient determined to be predisposed to the condition but not yet diagnosed as having the condition, (b) impeding the development of the condition, and/or (c) relieving the condition, i.e. , causing regression of the condition and/or relieving one or more symptoms of the condition.
- Treating or “treatment of a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results such as the reduction of symptoms.
- beneficial or desired clinical results include, but are not limited to: preventing infection of a patient at risk of RSV infection; or reducing the severity of infection by RSV, e.g., by reducing one or more symptoms, reducing the length of time of infection, etc.
- strain or "RSV strain” refers to any RSV. Strains include, but are not limited to, clinical isolates, variants, mutants, and the like. Strains may be palivizumab-resistant or palivizumab-sensitive. Strains may be of either RSV type, A or B. general, strains are distinguishable by one or more genetic mutations, even if such mutation does not confer a different characteristic to the virus.
- an "epitope region” is a sequence identified as including those amino acids that take part in the binding interaction with the antibody. For example, although certain assays have adequate resolution to identify a region of antigen amino acids within which antibody binding interactions take place, not all of the amino acids actually take part in the binding interaction.
- the “epitope”, as used herein, is the combination of amino acids within the epitope region that interact with the antibody to provide the specific binding between antigen and antibody.
- antibody construct refers to an antibody wherein at least a portion of the antibody is derived from an antibody from a human patient who had been exposed to the antigen of interest.
- An antibody construct may be an entire antibody or a fragment or portion thereof, provided the antibody, fragment, or portion has the recited affinity for F protein.
- An antibody construct may be fully human, humanized, or chimeric.
- An antibody construct may comprise amino acid sequences derived from a single patient, multiple patients, and/or known antibody sequences (e.g., a consensus constant region sequence).
- the term "antibody fragment” refers to any portion of the antibody that recognizes an epitope. Antibody fragments may be glycosylated.
- the antibody fragment may be a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a rlgG fragment, a functional antibody fragment, single chain recombinant forms of the foregoing, and the like.
- F(ab')2, Fab, Fab' and Fv are antigen-binding fragments that can be generated from the variable region of IgG and IgM. They vary in size, valency, and Fc content.
- the fragments may be generated by any method, including expression of the constituents (e.g., heavy and light chain portions) by a cell or cell line, or multiple cells or cell lines.
- F(ab')2 fragments contain two antigen-binding regions joined at the hinge through disulfide linkages and lack most of the Fc region.
- Fab fragments are derived from F(ab')2 but include only one antigen binding region. They may contain a small portion of Fc.
- Fv is the smallest fragment produced from IgG and IgM that contains a complete antigen-binding site. Fv comprises a portion of the variable heavy and light chains, held together by non-covalent interactions. These chains tend to dissociate upon dilution, but can be cross-linked, for example using glutaraldehyde, intermolecular disulfides or a peptide linker. Fv fragments have the same binding properties and similar three-dimensional binding characteristics as Fab.
- rlgG refers to reduced IgG or half-IgG, containing one heavy chain and one light chain. rlgG can be produced by selectively reducing the hinge-region disulfide bonds.
- the term "fully human antibody” refers to an antibody, antibody construct, or antibody fragment consisting entirely of human amino acid sequence. That is, the amino acid sequence of the human monoclonal antibody construct is derived from a human cell. This may be obtained in different ways.
- the human monoclonal antibody construct consisting of human amino acid sequence can be obtained from a cell engineered to express the variable region heavy and light chains and/or CDRs from an antibody derived from a human patient (e.g., a patient who had been exposed to RSV and/or RSV F protein).
- the human monoclonal antibody construct can be obtained from a hybridoma, wherein the B-cell is a human B-cell.
- the antibody construct or antibody fragment binds to F protein with a higher affinity than palivizumab. In one embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of between about 2: 1 and about 500: 1 versus palivizumab. In a preferred embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of between about 20: 1 and about 200: 1 versus palivizumab. It is to be understood that the ratio can be ranges between any two of these values, or any value there between (including endpoints). In one embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of at least about 2: 1 versus palivizumab.
- the antibody construct or antibody fragment binds to F protein with an affinity of at least about 5: 1 versus palivizumab. In one embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of at least about 10: 1 versus palivizumab. In one embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of at least about 50: 1 versus palivizumab. In one embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of at least about 100: 1 versus palivizumab. In one embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of at least about 200: 1 versus palivizumab.
- the antibody construct or antibody fragment binds to F protein with an affinity of at least about 300: 1 versus palivizumab. In one embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of at least about 400: 1 versus palivizumab. In one embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of at least about 500: 1 versus palivizumab.
- the antibody construct or antibody fragment is capable of neutralizing RSV virus strains. In one embodiment, the antibody construct or antibody fragment is capable of neutralizing at least one RSV strain. In one embodiment, the antibody construct or antibody fragment is capable of neutralizing at least one RSV type A strain. In one embodiment, the antibody construct or antibody fragment is capable of neutralizing at least one RSV type B strain. In a preferred embodiment, the antibody construct or antibody fragment is capable of neutralizing at least one RSV type A strain and at least one type B strain. In an especially preferred embodiment, the antibody construct or antibody fragment is capable of neutralizing one or more strains that are resistant to palivizumab.
- the antibody construct or antibody fragment is capable of neutralizing at least one RSV strain with a greater neutralization capacity than palivizumab.
- the neutralization capacity of the antibody construct or antibody fragment against at least one RSV strain is at least two times greater than the neutralization capacity of palivizumab.
- the neutralization capacity of the antibody construct or antibody fragment against at least one RSV strain is at least five times greater than the neutralization capacity of palivizumab.
- the neutralization capacity of the antibody construct or antibody fragment against at least one RSV strain is at least ten times greater than the neutralization capacity of palivizumab.
- the neutralization capacity of the antibody construct or antibody fragment against at least one RSV strain is at least fifteen times greater than the neutralization capacity of palivizumab.
- the antibody construct or antibody fragment comprises an amino acid sequence having at least 85% sequence homology to the amino acid sequence of SEQ ID NO.: 1. In one embodiment, the antibody construct or antibody fragment comprises an amino acid sequence having at least 90% sequence homology to the amino acid sequence of SEQ ID NO.: 1. In one embodiment, the antibody construct or antibody fragment comprises an amino acid sequence having at least 95% sequence homology to the amino acid sequence of SEQ ID NO.: 1. In one embodiment, the antibody construct or antibody fragment comprises an amino acid sequence having at least 96% sequence homology to the amino acid sequence of SEQ ID NO. : 1. In one embodiment, the antibody construct or antibody fragment comprises an amino acid sequence having at least 97% sequence homology to the amino acid sequence of SEQ ID NO.: 1.
- the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 85% sequence homology to the amino acid sequence of SEQ ID NO.: 8, SEQ ID NO.: 9, and/or SEQ ID NO.: 10. In one embodiment, the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 90% sequence homology to the amino acid sequence of SEQ ID NO.: 8, SEQ ID NO.: 9, and/or SEQ ID NO.: 10. In one embodiment, the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 95% sequence homology to the amino acid sequence of SEQ ID NO.: 8, SEQ ID NO.: 9, and/or SEQ ID NO.: 10.
- the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 96% sequence homology to the amino acid sequence of SEQ ID NO.: 8, SEQ ID NO.: 9, and/or SEQ ID NO.: 10. In one embodiment, the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 97% sequence homology to the amino acid sequence of SEQ ID NO.: 8, SEQ ID NO.: 9, and/or SEQ ID NO.: 10. In one embodiment, the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 98% sequence homology to the amino acid sequence of SEQ ID NO.: 8, SEQ ID NO.: 9, and/or SEQ ID NO.: 10.
- the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 99% sequence homology to the amino acid sequence of SEQ ID NO.: 8, SEQ ID NO.: 9, and/or SEQ ID NO.: 10. In one embodiment, the antibody construct or antibody fragment comprises at least one CDR with the amino acid sequence of SEQ ID NO.: 8, SEQ ID NO.: 9, and/or SEQ ID NO.: 10. In a preferred embodiment, the antibody construct or antibody fragment comprises a heavy chain CDRl with the amino acid sequence of SEQ ID NO.: 8, a heavy chain CDR2 with the amino acid sequence of SEQ ID NO.: 9, and a heavy chain CDR3 with the amino acid sequence of SEQ ID NO. : 10.
- the antibody construct or antibody fragment comprises an amino acid sequence having at least 85% sequence homology to the amino acid sequence of SEQ ID NO.: 3. In one embodiment, the antibody construct or antibody fragment comprises an amino acid sequence having at least 90% sequence homology to the amino acid sequence of SEQ ID NO.: 3. In one embodiment, the antibody construct or antibody fragment comprises an amino acid sequence having at least 95% sequence homology to the amino acid sequence of SEQ ID NO.: 3. In one embodiment, the antibody construct or antibody fragment comprises an amino acid sequence having at least 96% sequence homology to the amino acid sequence of SEQ ID NO.: 3. In one embodiment, the antibody construct or antibody fragment comprises an amino acid sequence having at least 97% sequence homology to the amino acid sequence of SEQ ID NO.: 3. In one embodiment, the antibody construct or antibody fragment comprises an amino acid sequence having at least 98% sequence homology to the amino acid sequence of SEQ ID NO.: 3. In one
- the antibody construct or antibody fragment comprises an amino acid sequence having at least 99% sequence homology to the amino acid sequence of SEQ ID NO.: 3. In one embodiment, the antibody construct or antibody fragment comprises the amino acid sequence of SEQ ID NO.: 3. In a preferred embodiment, the light chain variable region of the antibody construct or antibody fragment comprises the amino acid sequence of SEQ ID NO.: 3.
- the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 85% sequence homology to the amino acid sequence of SEQ ID NO.: 14, SEQ ID NO.: 15, and/or SEQ ID NO.: 16 In one embodiment, the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 90% sequence homology to the amino acid sequence of SEQ ID NO.: 14, SEQ ID NO.: 15, and/or SEQ ID NO.: 16. In one embodiment, the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 95% sequence homology to the amino acid sequence of SEQ ID NO.: 14, SEQ ID NO.: 15, and/or SEQ ID NO.: 16 .
- the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 96% sequence homology to the amino acid sequence of SEQ ID NO.: 14, SEQ ID NO.: 15, and/or SEQ ID NO.: 16. In one embodiment, the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 97% sequence homology to the amino acid sequence of SEQ ID NO.: 14, SEQ ID NO.: 15, and/or SEQ ID NO.: 16. In one embodiment, the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 98% sequence homology to the amino acid sequence of SEQ ID NO.: 14, SEQ ID NO.: 15, and/or SEQ ID NO.: 16.
- the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 99% sequence homology to the amino acid sequence of SEQ ID NO.: 14, SEQ ID NO.: 15, and/or SEQ ID NO.: 16. In one embodiment, the antibody construct or antibody fragment comprises at least one CDR with the amino acid sequence of SEQ ID NO.: 14, SEQ ID NO.: 15, and/or SEQ ID NO.: 16. In a preferred embodiment, the antibody construct or antibody fragment comprises a light chain CDR1 with the amino acid sequence of SEQ ID NO.: 14, a light chain CDR2 with the amino acid sequence of SEQ ID NO.: 15, and a light chain CDR3 with the amino acid sequence of SEQ ID NO.: 16.
- the invention further relates to derivatives of the antibody construct or antibody fragment described herein.
- derivative encompasses any mutants of the antibody construct differing by the addition, deletion, and/ or substitution of at least one amino acid.
- the derivative is a mutant of the antibody construct that carries at least one conservative substitution in any of the CDRs in the heavy chain and/or light chain as indicated in Figures 4 and 5. More preferably, the mutant has not more than 5, not more than 4, preferably not more than 3, particularly preferred not more than 2 conservative substitutions.
- the capacity of the fragment or derivative of the antibody to bind to the epitope can be determined by direct ELISA, for example, as described in the Examples section below.
- the term "conservative substitution” means a replacement of one amino acid belonging to a particular physico- chemical group with an amino acid belonging to the same physico-chemical group.
- the physico-chemical groups are defined as follows:
- the group of non-polar amino acids comprises: glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, and tryptophan.
- the group of amino acids having uncharged polar side chains comprises asparagine, glutamine, tyrosine, cysteine, and cysteine.
- the physico-chemical group of amino acids having a positively charged polar side chain comprises lysine, arginine, and histidine.
- the physico-chemical group of amino acids having a negatively charged polar side chain comprises aspartic acid and glutamic acid, also referred to as aspartate and glutamate.
- the light chain of the antibody construct or antibody fragment according to the present invention is of the kappa or lambda type.
- the light chain is of the kappa type.
- the light chain may be either a naturally occurring chain including a naturally rearranged, a genetically modified or synthetic type of light chain.
- the heavy chain of the antibody of the present invention is selected from all human isotypes, namely IgM, IgA, or IgG.
- the light chain and heavy chain may either be covalently linked as a single-chain antibody (e. g. bivalent scFv, bifunctional scFv and bispecific scFv) or non-covalently linked with each other.
- the antibody construct or antibody fragment recognizes an epitope on the F protein of RSV.
- the epitope is within antigenic region WA.
- the antibody construct recognizes an epitope within antigenic region WA having at least 85% sequence homology to a portion of the amino acid sequence of SEQ ID NO.: 23 or SEQ ID NO.: 24.
- the antibody construct recognizes an epitope within antigenic region II/A having at least 90% sequence homology to a portion of the amino acid sequence of SEQ ID NO.: 23 or SEQ ID NO.: 24.
- the antibody construct recognizes an epitope within antigenic region WA having at least 95% sequence homology to a portion of the amino acid sequence of SEQ ID NO.: 23 or SEQ ID NO.: 24. In one embodiment, the antibody construct recognizes an epitope within antigenic region II/A having at least sequence 96% homology to a portion of the amino acid sequence of SEQ ID NO.: 23 or SEQ ID NO.: 24. In one embodiment, the antibody construct recognizes an epitope within antigenic region II/A having at least 97% sequence homology to a portion of the amino acid sequence of SEQ ID NO.: 23 or SEQ ID NO.: 24.
- the antibody construct recognizes an epitope within antigenic region WA having at least 98% sequence homology to a portion of the amino acid sequence of SEQ ID NO.: 23 or SEQ ID NO.: 24. In one embodiment, the antibody construct recognizes an epitope within antigenic region II/A having at least 99% sequence homology to a portion of the amino acid sequence of SEQ ID NO.: 23 or SEQ ID NO.: 24. [0084] In one embodiment, the antibody construct recognizes an epitope within antigenic region II/ A having at least 85% sequence homology to a portion of the amino acid sequence of SEQ ID NO. : 25 or SEQ ID NO.: 26.
- the antibody construct recognizes an epitope within antigenic region 11/ A having at least 90% sequence homology to a portion of the amino acid sequence of SEQ ID NO.: 25 or SEQ ID NO.: 26. In one embodiment, the antibody construct recognizes an epitope within antigenic region 11/ A having at least 95% sequence homology to a portion of the amino acid sequence of SEQ ID NO.: 25 or SEQ ID NO.: 26. In one embodiment, the antibody construct recognizes an epitope within antigenic region 11/ A having at least sequence 96% homology to a portion of the amino acid sequence of SEQ ID NO.: 25 or SEQ ID NO.: 26.
- the antibody construct recognizes an epitope within antigenic region WA having at least 97% sequence homology to a portion of the amino acid sequence of SEQ ID NO.: 25 or SEQ ID NO.: 26. In one embodiment, the antibody construct recognizes an epitope within antigenic region 11/ A having at least 98% sequence homology to a portion of the amino acid sequence of SEQ ID NO.: 25 or SEQ ID NO.: 26. In one embodiment, the antibody construct recognizes an epitope within antigenic region 11/ A having at least 99% sequence homology to a portion of the amino acid sequence of SEQ ID NO.: 26 or SEQ ID NO.: 26.
- the present invention further relates to nucleotide sequences encoding the antibody construct or antibody fragment, or portions thereof.
- the nucleotide sequence comprises a cDNA sequence.
- the nucleotide sequence comprises a cDNA sequence encoding for the heavy chain variable region.
- the cDNA sequence encoding for the heavy chain variable region has at least 85% sequence homology to the nucleotide sequence of SEQ ID NO.: 2.
- the cDNA sequence encoding for the heavy chain variable region has at least 90% sequence homology to the nucleotide sequence of SEQ ID NO.: 2.
- the cDNA sequence encoding for the heavy chain variable region has at least 95% sequence homology to the nucleotide sequence of SEQ ID NO.: 2.
- the cDNA sequence encoding for the heavy chain variable region has at least 96% sequence homology to the nucleotide sequence of SEQ ID NO.: 2.
- the cDNA sequence encoding for the heavy chain variable region has at least 97% sequence homology to the nucleotide sequence of SEQ ID NO.: 2. In one embodiment, the cDNA sequence encoding for the heavy chain variable region has at least 98% sequence homology to the nucleotide sequence of SEQ ID NO.: 2. In one
- the cDNA sequence encoding for the heavy chain variable region has at least 99% sequence homology to the nucleotide sequence of SEQ ID NO.: 2.
- the nucleotide sequence comprises a cDNA sequence encoding for the light chain variable region.
- the cDNA sequence encoding for the light chain variable region has at least 85% sequence homology to the nucleotide sequence of SEQ ID NO.: 4.
- the cDNA sequence encoding for the light chain variable region has at least 90% sequence homology to the nucleotide sequence of SEQ ID NO. : 4.
- the cDNA sequence encoding for the light chain variable region has at least 95% sequence homology to the nucleotide sequence of SEQ ID NO.: 4.
- the cDNA sequence encoding for the light chain variable region has at least 96% sequence homology to the nucleotide sequence of SEQ ID NO.: 4. In one embodiment, the cDNA sequence encoding for the light chain variable region has at least 97% sequence homology to the nucleotide sequence of SEQ ID NO.: 4. In one embodiment, the cDNA sequence encoding for the light chain variable region has at least 98% sequence homology to the nucleotide sequence of SEQ ID NO.: 4. In one embodiment, the cDNA sequence encoding for the light chain variable region has at least 99% sequence homology to the nucleotide sequence of SEQ ID NO.: 4.
- the nucleotide sequence encodes for one or more CDRs.
- the nucleotide sequence encoding for one or more CDRs comprises a nucleotide with at least 85% sequence homology to the nucleotide sequence of SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 11, SEQ ID NO.: 12, and/or SEQ ID NO.: 13.
- the nucleotide sequence encoding for one or more CDRs comprises a nucleotide with at least 90% sequence homology to the nucleotide sequence of SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 11, SEQ ID NO.: 12, and/or SEQ ID NO.: 13.
- the nucleotide sequence encoding for one or more CDRs comprises a nucleotide with at least 96%, 97%, 98%, or 99% sequence homology to the nucleotide sequence of SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 11, SEQ ID NO.: 12, and/or SEQ ID NO.: 13.
- the nucleotide sequence encoding for one or more CDRs comprises the nucleotide sequence of SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 11, SEQ ID NO.: 12, and/or SEQ ID NO.: 13.
- the nucleotide sequence comprising the nucleotide sequence of SEQ ID NO.: 5, SEQ ID NO.: 6, and/or SEQ ID NO.: 7 encodes for a CDR of the heavy chain variable region of the antibody construct or antibody fragment. In one embodiment, the nucleotide sequence comprising the nucleotide sequence of SEQ ID NO.: 5, SEQ ID NO.: 6, and/or SEQ ID NO.: 7 encodes for a CDR of the light chain variable region of the antibody construct or antibody fragment.
- the nucleotide sequence comprising the nucleotide sequence of SEQ ID NO.: 11, SEQ ID NO.: 12, and/or SEQ ID NO.: 13 encodes for a CDR of the heavy chain variable region of the antibody construct or antibody fragment.
- the nucleotide sequence comprising the nucleotide sequence of SEQ ID NO.: 11, SEQ ID NO.: 12, and/or SEQ ID NO.: 13 encodes for a CDR of the light chain variable region of the antibody construct or antibody fragment.
- the nucleotide sequence is a cDNA sequence.
- the nucleotide sequence is an RNA molecule encoded by a nucleotide comprising SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 11, SEQ ID NO.: 12, and/or SEQ ID NO.: 13.
- the nucleotide sequence is an RNA molecule encoded by a nucleotide comprising SEQ ID NO.: 2.
- the nucleotide sequence is an RNA molecule encoded by a nucleotide comprising SEQ ID NO.: 4.
- the present invention further provides vectors comprising at least one nucleic acid encoding the light chain of the antibody construct or antibody fragment of the present invention and/or at least one nucleic acid encoding the heavy chain of the antibody construct or antibody fragment of the present invention.
- the nucleic acids may be either present in the same vector or may be present in the form of binary vectors.
- the vector preferably comprises the promoter operatively linked to the nucleic acid in order to facilitate expression of the nucleic acid encoding the light and/or heavy chain.
- the vector also includes an origin for replication and maintenance in a host cell.
- the vector may also comprise a nucleotide sequence encoding a signal sequence located 5' of the nucleic acid encoding the light chain and/or heavy chain.
- the signal sequence may facilitate secretion of the encoded chain into the medium.
- the vector may further comprise a His-tag coding nucleotide sequence resulting in the expression of a construct for producing a fusion product with a His-tag at the N-terminus of the light and/ or heavy chain of the antibody construct or antibody fragment, which facilitates purification of the protein.
- the antibody construct or antibody fragment according to the present invention is modified.
- the modifications include the di-, oligo-, or polymerization of the monomeric form e. g. by cross-linking using
- dicyclohexylcarbodiimide The di-, oligo-, or polymers can be separated from each other by gel filtration. Further modifications include side chain modifications, e. g. modifications of ⁇ -amino-lysine residues, or amino and carboxy-terminal modifications, respectively.
- Further modifications include post-translational modifications, e.g. glycosylation and/or partial or complete deglycosylation of the protein, and disulfide bond formation.
- the antibody construct or fragment may also be conjugated to a label, such as an enzymatic, fluorescent or radioactive label.
- the antibody construct or antibody fragment is lyophilized.
- this invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising the antibody construct or antibody fragment as described herein.
- Such compositions include various excipients, diluents, carriers, and such other inactive agents well known to the skilled artisan.
- the pharmaceutical composition comprises the antibody construct or antibody fragment and a pharmaceutically acceptable carrier, diluent, or excipient.
- This invention further relates to methods and composition for making the antibody construct or antibody fragment as described herein.
- the human B-cell may be fused to a myeloma or heteromyeloma to produce a hybridoma in accordance with known techniques according to the classical Kohler and Milstein approach, as described by Lang et al "Prophylaxis and therapy of Paseudomonas aeruginosa infection in cystic fibrosis and immunocompromised patients" Vaccine 22: S44-S48 (2004), which is incorporated herein by reference in its entirety.
- the B cell is cultured and the cDNA sequence of a heavy chain variable region, light chain variable region, and/or one or more CDRs is isolated therefrom.
- the cDNA sequences can be used to generate one or more vectors.
- the vector(s) can be introduced into a cell such that the cell produces the antibody construct, antibody fragment, or portion thereof.
- the cell may be a prokaryotic cell or a eukaryotic cell.
- the cell is a plant cell or a mammalian cell.
- the cell is a human cell.
- the cell is a HEK293 cell.
- the cell is a PerC6 cell, a CHO cell, a COS cell, or a HELA cell.
- the antibody construct or antibody fragment described herein is produced in a plant cell.
- Methods of producing antibodies in plant cells are described, for example, in U.S. Patent Nos. 8,119,406 and 8,513,397, each of which is incorporated herein by reference in its entirety.
- the cell is from a tobacco plant (e.g., genus Nicotiana). Benefits can be realized from expression in a plant host system. For example, the alternate glycosylations of a plant system can improve pharmacokinetics and/or desirable cytotoxic effects of the product antibody.
- the host cell comprises at least one nucleic acid encoding the light chain and at least one nucleic acid encoding the heavy chain and is capable of assembling the antibody construct such that a 3-dimensional structure is generated which is equivalent to the 3-dimensional structure of a human antibody produced by a human B-cell. If the light chain is produced separately from the heavy chain, then both chains may be purified and subsequently be assembled to produce an antibody having essentially the 3- dimensional structure of a human antibody as produced by a human B-cell.
- the host cell comprises at least one nucleic acid encoding at least the antigen-binding portion of the light chain and at least one nucleic acid encoding at least the antigen-binding portion of the heavy chain, and is capable of assembling the antibody construct or fragment such that the antibody construct or fragment is capable of binding the antigen.
- the antibody construct or antibody fragment may also be obtained by recombinant expression of the encoded light and/or heavy chain (or portion thereof), wherein the nucleic acid is produced by isolating a nucleic acid encoding a human antibody and grafting of the nucleic acid sequence encoding the CDRs as defined in the figures onto the isolated nucleic acid.
- Antibodies or antibody fragments can be purified, for example from cell culture supernatant, by any method. Exemplary methods of antibody purification are described in Liu et al., "Recovery and purification process development for monoclonal antibody production,” mAbs 2(5): 480-499 (2010), which is incorporated herein by reference in its entirety.
- this invention relates to a chromatography column or membrane comprising an antibody construct or antibody fragment as described herein, wherein the antibody construct or antibody fragment is bound to the chromatography column or membrane.
- the chromatography column or membrane comprises Protein A, e.g. a Protein A resin.
- the chromatography column or membrane comprises an ion exchange resin.
- the chromatography column or membrane comprises a hydrophobic charge induction chromatography column.
- the descriptions and examples herein are directed to production of the antibody construct or fragment within a single cell or cell line, it is contemplated that one or more portions of the antibody construct or fragment may be produced by separate cells or cell lines and then combined to form a functional antibody construct or fragment.
- the heavy chain (or portion thereof) may be produced by one cell, and the light chain (or portion thereof) may be produced by a second cell.
- the components may be isolated or purified, e.g. from the cell culture supernatant, and covalently or non-covalently linked by routine methods.
- this invention relates to a method for determining the potency of a putative antibody to strains of RSV that are resistant to commercially available antibodies, e.g., palivizumab.
- the binding affinity, neutralizing capacity, antigenicity, or any other measure of the usefulness of the putative antibody against at least one RSV strain may be compared to the antibody construct of the current invention.
- a putative antibody that is determined to bind with an affinity equal to or greater than, have a neutralization capacity equal to or greater than, and/or exhibit equal or less antigenicity than, an antibody construct as described herein is further tested for effectiveness against RSV.
- This invention further relates to methods of treatment using the antibody construct or antibody fragment as described herein.
- this invention relates to methods of preventing RSV infection in a patient at risk of infection by RSV.
- upper respiratory infection is prevented.
- lower respiratory infection is prevented.
- both upper and lower respiratory infection is prevented.
- this invention relates to methods of treating RSV infection in a patient.
- upper respiratory infection is treated.
- lower respiratory infection is treated.
- both upper and lower respiratory infection is treated.
- the patient is a human.
- the patient is an infant (e.g., under 12 months of age).
- the patient is a human having a condition that increases the risk of RSV infection.
- Conditions that increase the risk of RSV infection include, but are not limited to, being under 6 months of age; being under 12 months of age and born prematurely (e.g., before 40 weeks gestational age); lung disease; chronic obstructive pulmonary disease; congenital heart disease; congestive heart failure; weakened immune system; asthma; immunodeficiency (e.g., transplanted organ, leukemia, HIV/ AIDS).
- RSV infections are considered problematic mainly for (preterm) infants, also elderly and immune-compromised patients (e.g. HIV positive patients, patients under chemotherapy or patients with another severe underlying condition) are at risk.
- Palivizumab is not normally used to treat these patient groups due to low efficacy.
- AR-201 shows higher affinity for the virus, recognizes more viral strains and is currently being engineered for enhanced ADCC and prolonged half-life. Such improved potency and durability of action are expected to enhance the medicinal value of an anti-RSV mAb in immune-compromised and elderly patients infected with RSV.
- the antibody construct or antibody fragment is administered to a patient at risk of infection by RSV. In one embodiment, the antibody construct or antibody fragment is administered to a patient having or believed to have an infection by RSV. In one embodiment, a therapeutically effective amount of the antibody construct or antibody fragment is administered. In one embodiment, the therapeutically effective amount is between about 1 mg per kg body weight and about 1000 mg per kg body weight. In a preferred embodiment, the therapeutically effective amount is between about 1 mg per kg body weight and about 100 mg per kg body weight. In a more preferred embodiment, the therapeutically effective amount is between about 5 mg per kg body weight and about 50 mg per kg body weight. It is to be understood that the amount can be a range between any two of these values, or any value there between (including endpoints).
- the antibody construct or antibody fragment may be administered by any appropriate route.
- the compositions, provided herein or known, suitable for administration in accordance with the methods provide herein, can be suitable for a variety of delivery modes including, without limitation, intramuscular and intravenous delivery.
- Compositions suitable for internal, pulmonary, rectal, nasal, vaginal, lingual, intraarterial, intraperitoneal, intracutaneous and subcutaneous routes may also be used.
- Sustained release dosage forms may also be used. All dosage forms may be prepared using methods that are standard in the art (see e.g., Remington's Pharmaceutical Sciences, 16th ed., A. Oslo editor, Easton Pa. 1980).
- the antibody construct or fragment is administered multiple times. In a more preferred embodiment, the antibody construct or fragment is administered once a month during RSV season. Most preferably the antibody construct or fragment is administered only once per season. RSV season is generally November through April (in the Northern Hemisphere), but can be longer depending on the area and other factors (e.g., the primary strains that are circulating in a given year).
- the invention relates to a bag for intravenous delivery, comprising an IV bag containing the antibody construct or antibody fragment and a pharmaceutically acceptable excipient.
- Ig immunoglobulin
- Conditioned supernatant was generated by stimulating isolated peripheral blood lymphocytes with phytohaemagglutinin (PHA, 5 ⁇ g/mL) and phorbol myristate acetate (PMA, 10 ng/mL) for 36 hours, followed by removal of cells and debris prior to cryopreservation. The conditioned supernatant was thawed prior to addition to the RSV-EIA selected
- Positive wells were identified by colorimetric measurement, and cells from positive wells were expanded at low density in the presence of irradiated feeder cells and cell culture medium containing 10% FCS and 10% conditioned supernatant (as described above) over a period of several days. After retesting supernatants for specificity to RSV F protein, cells from positive wells were collected and processed for RNA isolation.
- RNA from selected B-cells was used to generate 5'RACE cDNA, followed by isotype specific RT-PCR to identify the variable region of the heavy chain (HC) and light chain (LC) as described by Welschof M, et al., 1995.
- the variable region of the HC was combined by PCR with the constant region of an IgGl which is essentially in accordance with the IMGT reference sequence Y14737 (Lefranc, M.-P. et al., 2001, The variable region of the HC was combined by PCR with the constant region of an IgGl which is essentially in accordance with the IMGT reference sequence Y14737 (Lefranc, M.-P. et al., 2001, The variable region of the HC was combined by PCR with the constant region of an IgGl which is essentially in accordance with the IMGT reference sequence Y14737 (Lefranc, M.-P. et al., 2001, The variable region of the HC was combined by PCR with
- Figures 2 (heavy chain, SEQ ID NO.: 1 and 2) and 3 (light chain, SEQ ID NO.: 3 and 4).
- the nucleotide and corresponding amino acid sequences of individual CDR regions are provided in Figures 4 (heavy chain: CDR1, SEQ ID NO.: 5 and 8; CDR2, SEQ ID NO.: 6 and 9; CDR3, SEQ ID NO.: 7 and 10) and 5 (light chain: CDR1, SEQ ID NO.: 11 and 14; CDR2, SEQ ID NO.: 12 and 15; CDR3, SEQ ID NO.: 13 and 16).
- the isolated and sequenced human antibody AR201 was tested for binding characteristics to RSV F protein, as shown in Figure 6.
- Hek293T cells were seeded at 300,000 cells per well of a six-well plate and cultivated overnight. After one day, medium was exchanged, and a freshly prepared solution of expression plasmids was added carefully to the cells. Cells were incubated for another 24 hours and medium was exchanged again for Pro293a-CDM medium. After three to five days, the resulting supernatant containing recombinant antibody was collected and antibody was purified by affinity chromatography with a HiTrap Protein A sepharose affinity column (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Resulting antibody was stored at -20 °C until use.
- ELISA plates were coated as described above, and serial dilutions of purified AR201 or an unrelated fully human monoclonal IgGl antibody (control) were added. After careful washing, bound antibodies were detected with anti-human IgG-HRP labeled secondary antibody.
- EC50 values were calculated based on colorimetric measurement of the bound antibody applying a variable slope-four parameter equation (GraphPad Prism Software V5.02, GraphPad Software Inc. San Diego, CA, US). The EC50 value of AR201 was calculated as 0.1031 nM. No EC50 value could be calculated for the control monoclonal human IgGl antibody.
- RSV-A Long ATCC VR-26
- RSV-B ATCC VR 1580
- RSV-A2 ATCC VR-1540
- LCG LGC Standards S.a.r.L, Molsheim, France
- the supernatant of positive wells was cryopreserved at -80 °C until further use.
- assay plates were coated with a polyclonal anti-RSV antibody at 500 ng/ml. Meanwhile, 10 ⁇ g/ml AR201 or human monoclonal IgGl isotype control antibody were incubated with RSV at a 1:10 dilution of the frozen amplified RSV strains.
- the percentage of neutralization for each concentration of the RSV-specific antibodies was calculated based on the number of plaques per well. Based on the concentration of the antibody stock, the minimal neutralizing antibody concentration achieving > 50% virus neutralization was calculated. As shown in Table 2, AR201 was at least twice as efficient in neutralizing viral particles of either strain and preventing the formation of syncytia indicative of an active, reproductive infection, as compared to palivizumab. An IgG isotype human monoclonal control antibody had no effect on the prevention of infections of Vero cells with either of the RSV reference strains.
- ELISA plates were coated with individual RSV isolates (diluted 1:10), and subsequently incubated with AR201, palivizumab, or a human monoclonal IgG isotype control antibody, at 1 ⁇ g/mL. A secondary anti-human IgG-HRP labeled antibody was added to each well.
- Bound human IgG was detected by colorimetric measurement.
- the background absorbance as determined by the signal of the control IgG monoclonal antibody, was deducted twice from the signal detected with AR201 or palivizumab and any absorbance value greater than 0.1 was considered a positive signal.
- the experiment was repeated three times; a representative experiment is shown in Figures 8A and 8B.
- AR201 recognized all 21 clinical isolates to varying extent, whereas palivizumab always showed a lesser signal intensity.
- the ratio of the binding signal of AR201 over palivizumab was analyzed. Any clinical isolate with a binding signal ratio of 5 or greater of AR201 over palivizumab was considered a palivizumab-resistant isolate.
- plaque reduction assay In further analysis, 68 additional isolates were analyzed by an alternative method: plaque reduction assay. This second assay gave a different output format compared to the first assay (ELISA): for isolate #1 and isolate #20 ("first, ELISA assay" a series of antibody dilutions were tested to find the IC50 which was considered for further testing. In the plaque reduction assay, analysis involved testing ONE antibody
- AR201 and palivizumab in the plaque reduction assay were incubated for 1 hr at 36°C, serially diluted and added to a nearly confluent monolayer of HEp-2 target cells in 24-well plates. After 1.5 hours the supernatant was aspirated, cells were overlayed with media and further incubated. Cytopathogenic effects were monitored daily and plates were fixed and stained after well- defined plaques had formed. Plaque forming units (PFUs ) were counted and reduction in PFUs by AR201 or palivizumab was calculated.
- PFUs Plaque forming units
- AR201 reduced the number of plaques in all cases to less than 1%, whereas palivizumab showed such strong reduction only on 45% of isolates.
- the ability to reduce plaques was on average 100-fold stronger for AR201 than for palivizumab.
- AR201 showed a 20-fold stronger reduction than palivizumab.
- AR201 fully inhibited plaque formation (sterilizing immunity) for 38 out of 42 tested A-strains (palivizumab: 2 out of 42 A strains), and for 23 out of 26 tested B-strains (palivizumab 7 out of 26 B-strains).
- aSterilizing immunity is defined as no plaques visible in the undiluted sample. For statistical purposes no plaques were assigned a value of 0.25 PFU. b Fisher's Exact Test, two-sided. °Number of isolates evaluated.
- AR201 and palivizumab was tested in an infectivity assay.
- the two RSV isolates were amplified on Vero cells and supernatant cryopreserved until further use.
- the dilution of the stock solution achieving a 50% infectivity rate in a tissue culture (TCID) was calculated based on the Spearman and Karber algorithm (Hierholzer et al., 1996, in Virology Methods Manual, edited by BMJ Mahy and HO Kangro, London: Academic Press, p.47).
- Virus stock were thawed and diluted to 10 x TCID50, and incubated with serial dilutions of antibodies AR201, palivizumab, or IgGl isotype human control monoclonal antibody.
- IC50 was calculated based on colorimetric measurement of the bound antibody by applying a variable slope-four parameter equation (GraphPad Prism Software V5.02, GraphPad Software Inc. San Diego, CA, US). The IC50 served as measure of the neutralization activity. IC50 is the antibody concentration (g/mL) that neutralizes the infectivity capacity of an RSV infective dose by 50%.
- Results are shown in Table 4.
- AR201 had an IC50 that was 18 times lower than palivizumab for isolate #1.
- For isolate #20 no neutralization capacity was observed for palivizumab, confirming that palivizumab does not bind to clinical isolate #20, whereas AR201 showed a similar IC50 on isolate #20 as seen with isolate #1.
- Ratio palivizumab/AR201 18.0 n/a
- GGGGCAMTMCMTGGAGTTGC [SEQ ID NO.: 22]). Amplified DNA was sequenced by Sanger sequencing at Microsynth (Balgach, Switzerland).
- a mutation at position 272 from lysine to another amino acid was described earlier as critical for loss of binding of palivizumab to the F protein (Zhu, et al., 2011; Adams et al., 2010), confirming the inability of palivizumab to bind within the epitope region of clinical isolate #20, confirming the sequence of isolate #20 region does not include an amino acid sequence functional as a palivizumab epitope. Further, two palivizumab-resistant mutant viruses were generated and both had a single amino acid (aa) mutation at position 262.
- Palivizumab-resistant mutants of RSV/A/Tracy and RSV/B/18537, respectively were generated after serial passage of the viruses in increasing concentrations (40, 80, 160 and 320 ⁇ g/mL) of palivizumab.
- Two palivizumab-resistant mutant viruses were isolated and both had a single amino acid (aa) mutation at position 262 as shown in Figure 10.
- Both parent strains had asparagine (an amide, neutral polar aa) at aa262 and both palivizumab resistant mutants had tyrosine (an aromatic neutral polar aa).
- the N262Y change occurred in the putative antigenic II domain and within the palivizumab defined epitope region (aa262-276).
- Palivizumab is the only anti-RSV monoclonal antibody that has been approved by the FDA to date for the prevention of RSV infection in infants and neonates. Palivizumab binds to antigenic region A on the F protein of RSV and inhibits fusion events of RSV with human lung epithelial cells.
- Efficient cross-inhibition of binding of two independent monoclonal antibodies can only occur if both antibodies recognize an epitope, or epitopes in close proximity (e.g., same epitope region), such that one antibody when bound to its epitope exerts steric hindrance for binding of the other antibody.
- AR201 reduced binding to AR201 coated plates to 22.5%, whereas pre-incubation with palivizumab affected binding to AR201 coated plates less and reduced the signal to only 29.2% of the control value.
- pre-incubation of RSV viral particles with AR201 reduced binding of RSV to palivizumab-coated plates to 40.8%, whereas pre-incubation with palivizumab reduced binding to palivizumab-coated plates at a slightly lower level, 35.3%.
- RSV F protein as palivizumab. That is, AR201 and palivizumab may have target epitopes in the similar region of amino acids on the RSV F-protein, but these antibodies may not necessarily interact with the same amino acid epitope members within the region. The difference in cross-inhibition indicates that AR201 recognizes an epitope similar to or close to the epitope of palivizumab, but not the identical epitope.
- Asp-N is an endoprotease which selectively cleaves peptide bonds N- terminal to aspartic acid residues. Asp-N digestion of the RSV F protein will cut the postulated palivizumab epitope N-terminal of amino acid positions 263 and 269, generating two protein fragments of 62 amino acids and 40 amino acids in length. Each peptide contains only a portion of the intact epitope for palivizumab, as shown in Figure 11. [0149] Purified recombinant F protein was incubated for 5 hours with Asp-N
- Palivizumab did not recognize any of the fragments of the complete digest of the RSV F protein, whereas AR201 still bound to as shown in Figure 10. This demonstrates that the two antibodies do not share the same epitope, and AR201 recognizes a combination of epitope amino acids in the 254-277 epitope region of the RSV F protein different from the amino acids making up the palivizumab target epitope amino acids. Furthermore, the epitope binding region exists in a three dimensional configuration such that AR201 binds differently than palivizumab.
- NSELLSLINDMPITNDQKK LMSSNVQIVRQQSYSI a segment of the F-Protein from RS-Virus (amino acids 254-288).
- DQKKLMSSN (SEQ ID NO: 29) but with different specificity. Small structural differences of the epitope, but also outside the epitope, affect the binding of both antibodies in completely different ways. Again, this suggests the binding interaction and epitope amino acids are different between AR201 and palivizumab.
- AR201 was produced in the plant Nicotiana benthamiana using the magnifection technology (Giritch et al, 2006 , Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors. Proceedings of the National Academy of Sciences of the United States of America 103(40): 14701-14706 ), which allows the production of antibodies within a very short timeframe.
- the use of plants with diminished fucosyl- and xylosyl-transferase (Strasser et al, 2008, Generation of glyco- engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure. Plant Biotechnology Journal 6(4):392-402.
- Agrobacterium tumefaciens Equal volumes of Agrobacterium cultures grown overnight were mixed in infiltration buffer (1 mM MES/10 mM MgS04 pH 5.5) resulting in a 1 : 1000 dilution for each individual culture.
- N. benthamiana plants grown for 4-5 weeks at 22°C with a 16/8 hr light/dark cycle were inverted and aerial parts were submerged in the bacterial/buffer solution. A vacuum of 0.5 bars was applied for 2 min and upon release the leaves got infiltrated with the bacterial solution.
- the mAb solution was polished via Q filtration (Mustang Acrodisc Q membrane; Pall), aliquoted and stored at -80°C until use.
- Antibody AR201 produced by CHO cells and alternatively in plants were compared with palivizumab in a Cotton Rat Model, as discussed below. Furthermore 2 AR201 mutants were produced in plants in order to increase the half-life of the antibody in serum.
- AR201-YTE is a triple mutation M252Y/S254T/T256E (YTE) which was introduced into the Fc portion.
- the Fc portion can be modified to present neonatal Fc receptor for IgG (FcRn) to enhance half-life.
- the antibody e.g., AR201
- the antibody comprise either or both of modified glycosylation (e.g., plant expression) and Fc mutations.
- Example 15 Effectiveness of IM Administration of AR201 and Palivizumab in the RSV/A/Tracv Cotton Rat Model
- Microneutralization assays were performed to test for neutralizing antibodies to RSV/ A/Tracy in the serum of cotton rats. Serum samples were heat inactivated at 56°C for 30 min, serially two-fold diluted in duplicates and mixed with plaque purified
- RSV/ A/Tracy (RSV/A). 96-well microtiter plates with nearly confluent HEp-2 cells were infected, further incubated and plaque formation was evaluated as described above
- the neutralizing antibody titer was defined as the serum dilution at which >50 % reduction in plaque formation was observed.
- RSV titers was tested in a cotton rat model.
- Cotton rats (A-F) received 5 mg antibody per kg bodyweight (or control formulation) on day - 1 and were challenged with RSV/ A/Tracy with 1.35 x 10 5 plaque forming units per cotton rat on day 0. Directly upon challenge blood was collected for determination of RSV-neutralizing antibody levels.
- On day +4 animals were sacrificed, blood samples were taken and the left lung lobe was removed. Blood was removed from lobe and it was transpleurally lavaged using 3 mL of Iscove's media with 15% glycerin mixed with 2% FBS-MEM (1 : 1, v:v). Lavage fluid was recovered by gently pressing the inflated lobe and used to lavage the right lobe following the same technique.
- RSV titers were determined as described above (example 8, "Plaque reduction assay).
- Both, AR201 and palivizumab reduced the virus titer in lung lavage fluid, however, while palivizumab reduced the titer only by a factor of ⁇ 70, AR201 reduced the lung virus titer over 500-fold and was thus several fold as effective as palivizumab.
- AR201 is a more potent monoclonal antibody in reducing lung virus titer compared to palivizumab in the RSV/A cotton rat model. See Figure 13.
- Example 16 Enhanced AR201.
- Different IgG mutations have been described to prolong the half-life of an
- FcRn neonatal Fc Receptor
- ADCC antibody dependent cell-mediated cytotoxicity
- ADCC is mediated by antibodies that crosslink antigen and immune cells e.g., natural killer cells, by binding at the same time to the epitope and to FcRIIIA (CD16a), the "ADCC-receptor", which is expressed on certain immune cells. This elicits ADCC, or killing of the target cells. Higher affinity of antibody to the FcyRIIIa translates to increased killing of the target by the immune cells, i.e. to better ADCC.
- the affinity of the antibody for the receptor can be influenced by altering the amino acid sequence of the antibody, but also by altering the glycosylation profile of the antibody (Shinkawa, J. Biol.
- Nicotiana benthamiana with the Magnifection system resulted in production of AR201 with hardly detectable fucose.
- the production of antibodies (e.g., AR201-YTE) with Fc modifications has shown good affinity and will enhance antibody half-life.
- An improved product can be an antibody with both of these features.
- an ant-RSV antibody (such as AR201) can be designed with Fc mutations for better half-life, and expressed in plants for better cytotoxicity. The combination can provide the efficacy beyond the individual contributions alone.
- Such an AR201 in the works, is an antibody which combines both effects of longer half-life by amino acid mutations as well as increased ADCC by optimized glycosylation profiles and/or amino acid mutations, and is expected to by superior to any anti-RSV described so far.
- improved anti-RSV antibodies can provide lifesaving advantages for infants, the elderly, and immunocompromised patients.
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pulmonology (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14/527,545 US9340604B1 (en) | 2014-10-29 | 2014-10-29 | Human monoclonal antibody specific for the F protein of respiratory syncytial virus (RSV) |
PCT/US2015/058076 WO2016069904A1 (en) | 2014-10-29 | 2015-10-29 | Human monoclonal antibody specific for the f protein of respiratory syncytial virus (rsv) |
Publications (3)
Publication Number | Publication Date |
---|---|
EP3212666A1 true EP3212666A1 (en) | 2017-09-06 |
EP3212666A4 EP3212666A4 (en) | 2018-06-27 |
EP3212666B1 EP3212666B1 (en) | 2021-04-28 |
Family
ID=55851920
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP15853881.9A Active EP3212666B1 (en) | 2014-10-29 | 2015-10-29 | Human monoclonal antibody specific for the f protein of respiratory syncytial virus (rsv) |
Country Status (6)
Country | Link |
---|---|
US (2) | US9340604B1 (en) |
EP (1) | EP3212666B1 (en) |
CN (1) | CN107207583B (en) |
CA (1) | CA2964633C (en) |
ES (1) | ES2875022T3 (en) |
WO (1) | WO2016069904A1 (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9340604B1 (en) * | 2014-10-29 | 2016-05-17 | Aridis Pharmaceuticals, Inc. | Human monoclonal antibody specific for the F protein of respiratory syncytial virus (RSV) |
CN111606992B (en) * | 2019-02-25 | 2022-08-19 | 天津昊免生物技术有限公司 | Fully human antibody for resisting respiratory syncytial virus |
WO2023025253A1 (en) * | 2021-08-25 | 2023-03-02 | 甘李药业股份有限公司 | Anti-rsv antibody and application thereof |
Family Cites Families (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5824307A (en) * | 1991-12-23 | 1998-10-20 | Medimmune, Inc. | Human-murine chimeric antibodies against respiratory syncytial virus |
US6818216B2 (en) * | 2000-11-28 | 2004-11-16 | Medimmune, Inc. | Anti-RSV antibodies |
US8119406B2 (en) | 2004-10-13 | 2012-02-21 | Protalix Ltd. | System and method for production of antibodies in plant cell culture |
TW200636064A (en) * | 2004-10-28 | 2006-10-16 | Centocor Inc | Anti-respiratory syncytial virus antibodies, antigens and uses thereof |
EP1997830A1 (en) | 2007-06-01 | 2008-12-03 | AIMM Therapeutics B.V. | RSV specific binding molecules and means for producing them |
JP5575377B2 (en) * | 2008-07-18 | 2014-08-20 | シスメックス株式会社 | RS virus detection kit and immunochromatography test device using anti-RS virus monoclonal antibody, and novel anti-RS virus monoclonal antibody |
WO2010025285A1 (en) | 2008-08-27 | 2010-03-04 | Arizona Board Of Regents For And On Behalf Of Arizona State University | A dna replicon system for high-level rapid production of vaccines and monoclonal antibody therapeutics in plants |
AU2010282508B2 (en) * | 2009-08-13 | 2015-05-28 | Crystal Bioscience Inc. | Transgenic animal for production of antibodies having minimal CDRs |
US8568726B2 (en) * | 2009-10-06 | 2013-10-29 | Medimmune Limited | RSV specific binding molecule |
CN102850454B (en) * | 2011-09-27 | 2014-06-25 | 上海博沃生物科技有限公司 | Anti-RSV (respiratory syncytial virus) human monoclonal antibody, and its preparation method |
US20130149300A1 (en) * | 2011-09-27 | 2013-06-13 | Icon Genetics Gmbh | MONOCLONAL ANTIBODIES WITH ALTERED AFFINITIES FOR HUMAN FCyRI, FCyRIIIa, AND C1q PROTEINS |
WO2013095091A2 (en) * | 2011-11-17 | 2013-06-27 | Aimm Therapeutics B.V. | Rsv g protein specific antibodies |
TWI659968B (en) * | 2013-03-14 | 2019-05-21 | 再生元醫藥公司 | Human antibodies to respiratory syncytial virus f protein and methods of use thereof |
US9340604B1 (en) * | 2014-10-29 | 2016-05-17 | Aridis Pharmaceuticals, Inc. | Human monoclonal antibody specific for the F protein of respiratory syncytial virus (RSV) |
-
2014
- 2014-10-29 US US14/527,545 patent/US9340604B1/en active Active
-
2015
- 2015-10-29 CA CA2964633A patent/CA2964633C/en active Active
- 2015-10-29 EP EP15853881.9A patent/EP3212666B1/en active Active
- 2015-10-29 US US15/523,190 patent/US10081671B2/en active Active
- 2015-10-29 CN CN201580059590.8A patent/CN107207583B/en active Active
- 2015-10-29 ES ES15853881T patent/ES2875022T3/en active Active
- 2015-10-29 WO PCT/US2015/058076 patent/WO2016069904A1/en active Application Filing
Also Published As
Publication number | Publication date |
---|---|
US20180030119A1 (en) | 2018-02-01 |
US10081671B2 (en) | 2018-09-25 |
CN107207583B (en) | 2021-08-17 |
US9340604B1 (en) | 2016-05-17 |
ES2875022T3 (en) | 2021-11-08 |
US20160122418A1 (en) | 2016-05-05 |
CA2964633A1 (en) | 2016-05-06 |
WO2016069904A1 (en) | 2016-05-06 |
CA2964633C (en) | 2024-01-23 |
CN107207583A (en) | 2017-09-26 |
EP3212666B1 (en) | 2021-04-28 |
EP3212666A4 (en) | 2018-06-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10196438B2 (en) | Human antibodies binding to RSV G protein | |
CA2908921C (en) | Human antibodies binding to rsv g protein | |
AU2008224259A1 (en) | Recombinant antibodies for treatment of respiratory syncytial virus infections | |
EP3220947B1 (en) | Antibodies that potently neutralize rabies virus and other lyssaviruses and uses thereof | |
US20170158753A1 (en) | Monoclonal antibodies directed against envelope glycoproteins from multiple filovirus species | |
CN116785427B (en) | Use of respiratory syncytial virus specific binding molecules | |
US10081671B2 (en) | Human monoclonal antibody specific for the F protein of respiratory syncytial virus (RSV) | |
CN116514960A (en) | Fully human monoclonal antibody of respiratory syncytial virus and application thereof | |
KR20150135231A (en) | Human Antibody Specific To Human Metapneumovirus, or Antigen-Binding Fragment Thereof | |
US11440951B2 (en) | Therapeutic antibodies to Marburg virus |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE |
|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE |
|
17P | Request for examination filed |
Effective date: 20170517 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
AX | Request for extension of the european patent |
Extension state: BA ME |
|
DAV | Request for validation of the european patent (deleted) | ||
DAX | Request for extension of the european patent (deleted) | ||
RIC1 | Information provided on ipc code assigned before grant |
Ipc: C07K 16/10 20060101AFI20180213BHEP |
|
A4 | Supplementary search report drawn up and despatched |
Effective date: 20180530 |
|
RIC1 | Information provided on ipc code assigned before grant |
Ipc: C07K 16/10 20060101AFI20180524BHEP |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: EXAMINATION IS IN PROGRESS |
|
17Q | First examination report despatched |
Effective date: 20200323 |
|
GRAP | Despatch of communication of intention to grant a patent |
Free format text: ORIGINAL CODE: EPIDOSNIGR1 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: GRANT OF PATENT IS INTENDED |
|
INTG | Intention to grant announced |
Effective date: 20201113 |
|
GRAS | Grant fee paid |
Free format text: ORIGINAL CODE: EPIDOSNIGR3 |
|
GRAA | (expected) grant |
Free format text: ORIGINAL CODE: 0009210 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE PATENT HAS BEEN GRANTED |
|
AK | Designated contracting states |
Kind code of ref document: B1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
REG | Reference to a national code |
Ref country code: GB Ref legal event code: FG4D |
|
REG | Reference to a national code |
Ref country code: CH Ref legal event code: EP |
|
REG | Reference to a national code |
Ref country code: AT Ref legal event code: REF Ref document number: 1386962 Country of ref document: AT Kind code of ref document: T Effective date: 20210515 |
|
REG | Reference to a national code |
Ref country code: DE Ref legal event code: R096 Ref document number: 602015068789 Country of ref document: DE |
|
REG | Reference to a national code |
Ref country code: IE Ref legal event code: FG4D |
|
RAP4 | Party data changed (patent owner data changed or rights of a patent transferred) |
Owner name: ARIDIS PHARMACEUTICALS, INC. |
|
REG | Reference to a national code |
Ref country code: DE Ref legal event code: R081 Ref document number: 602015068789 Country of ref document: DE Owner name: ARIDIS PHARMACEUTICALS, INC., LOS GATOS, US Free format text: FORMER OWNER: ARIDIS PHARMACEUTICALS, INC., SAN JOSE, CALIF., US |
|
REG | Reference to a national code |
Ref country code: LT Ref legal event code: MG9D |
|
REG | Reference to a national code |
Ref country code: AT Ref legal event code: MK05 Ref document number: 1386962 Country of ref document: AT Kind code of ref document: T Effective date: 20210428 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: LT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210428 Ref country code: NL Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210428 Ref country code: FI Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210428 Ref country code: BG Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210728 Ref country code: AT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210428 Ref country code: HR Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210428 |
|
REG | Reference to a national code |
Ref country code: ES Ref legal event code: FG2A Ref document number: 2875022 Country of ref document: ES Kind code of ref document: T3 Effective date: 20211108 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: LV Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210428 Ref country code: IS Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210828 Ref country code: GR Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210729 Ref country code: PT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210830 Ref country code: NO Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210728 Ref country code: PL Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210428 Ref country code: SE Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210428 Ref country code: RS Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210428 |
|
REG | Reference to a national code |
Ref country code: NL Ref legal event code: MP Effective date: 20210428 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: SK Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210428 Ref country code: SM Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210428 Ref country code: EE Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210428 Ref country code: CZ Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210428 Ref country code: DK Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210428 Ref country code: RO Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210428 |
|
REG | Reference to a national code |
Ref country code: DE Ref legal event code: R097 Ref document number: 602015068789 Country of ref document: DE |
|
PLBE | No opposition filed within time limit |
Free format text: ORIGINAL CODE: 0009261 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT |
|
26N | No opposition filed |
Effective date: 20220131 |
|
REG | Reference to a national code |
Ref country code: CH Ref legal event code: PL |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: IS Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210828 Ref country code: AL Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210428 |
|
REG | Reference to a national code |
Ref country code: BE Ref legal event code: MM Effective date: 20211031 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: MC Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210428 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: LU Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20211029 Ref country code: BE Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20211031 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: LI Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20211031 Ref country code: CH Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20211031 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: IE Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20211029 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: HU Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO Effective date: 20151029 |
|
P01 | Opt-out of the competence of the unified patent court (upc) registered |
Effective date: 20230404 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: CY Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210428 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: GB Payment date: 20231024 Year of fee payment: 9 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: ES Payment date: 20231110 Year of fee payment: 9 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: IT Payment date: 20231024 Year of fee payment: 9 Ref country code: FR Payment date: 20231026 Year of fee payment: 9 Ref country code: DE Payment date: 20231027 Year of fee payment: 9 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: MK Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20210428 |